Enzymes are biocatalysts which drive chemical reactions (metabolism) in vivo. Enzymes are primarily constructed by protein. There are enzymes which consist of protein alone, but most of the enzymes require components (cofactors) other than protein to express catalytic activity or enhance catalytic activity.
Enzymes have the following features:
(1) enzymes perform catalytic reactions under moderate conditions such as ordinary temperature and ordinary pressure; and
(2) enzymes have both “substrate specificity” to react only with a specific substrate (a substance subjected to the action of the enzyme), and “reaction specificity” to catalyze a specific chemical reaction without side reactions.
Enzymes which catalyze oxidoreduction in vivo are called as “oxidoreductases”. Among the enzymes, oxidoreductases which oxidize substrates using oxygen as an electron acceptor are particularly called as “oxidases”, and which reduce substrates are particularly called “reductases”. When a certain oxidoreductase is immobilized on the surface of an electrode, only a specific redox reaction selectively proceeds on the electrode surface by the catalytic action of the enzyme, and the change in the substance concerned in the redox reaction can be converted to an electrical signal by the electrode. Such an electrode is called “enzymatic electrode” and has been utilized as the electrode for various biosensors, fuel cells and so on.
In order to use an enzyme as an electrocatalyst, the enzyme needs to be immobilized on the surface of an appropriate carrier. However, since enzymes are soluble in water in general, there occurs a problem that the enzyme easily dissolves during use.
In addition, each enzyme has a part (active center) to which a substrate is specifically bound and where the catalytic reaction proceeds. Since the active center is often located deep in a complex three-dimensional structure of the protein molecule, it is difficult to perform a direct transfer of electron between the active center and the electrode. In this case, a low-molecular substance which gets into the active site of the enzyme, transfers electron with enzyme, and carries electron to the electrode is generally used together. Such a low-molecular substance is called “mediator”. However, since the electron transfer rate between enzyme and mediator depends on their molecular motion, the electron transfer rate is not necessarily rapid enough, limiting the current density of the enzymatic electrode.
In order to solve this problem, various ideas have been proposed.
For example, Patent Literature 1 discloses a nitrogen-containing carbonaceous composite material comprising a porous body made of a nitrogen-containing carbonaceous material having skeleton structure formed by carbon atoms and nitrogen atoms, and an oxidoreductase supported by the porous body.
The following is mentioned in Patent Literature 1:
(1) association between protein and carrier can be strengthened in such a manner that new bonds such as hydrogen bonds are generated between polar portions of the nitrogen-containing carbonaceous material and hydrophilic groups on the surface of the protein when the oxidoreductase is supported by the nitrogen-containing carbonaceous material, because the polar portions are scattered on the porous surface of the nitrogen-containing carbonaceous material; and
(2) examples of the oxidoreductase to be supported on a carrier are laccase, diaphorase, lipoxyamide dehydrogenase, alcohol dehydrogenase, glucose oxidase and oxidases which use sugars other than glucose as substrate, and glucose dehydrogenase and dehydrogenases which use sugars other than glucose as substrate.
Also, Patent Literature 2 discloses an enzymatic electrode comprising a conductive member, an enzyme, a first mediator and a second mediator, wherein the first mediator and the second mediator with different oxidoreduction potentials are immobilized on the conductive member by a carrier.
The following is mentioned in Patent Literature 2:
(1) density of the supported enzyme per effective surface area of the conductive member can be raised by the constitution above being employed;
(2) a second mediator, which transfers charge between the first mediator and the conductive member, is used in addition to the first mediator, which can quickly transfer electron with the enzyme, so that the enzymatic electrode can quickly transfer electron with the enzyme; and
(3) examples of the enzyme are glucose oxidase, galactose oxidase, bilirubin oxidase, pyruvate oxidase, D-amino-acid oxidase, L-amino-acid oxidase, amine oxidase, cholesterol oxidase, ascorbic acid oxidase, cytochrome oxidase, alcohol dehydrogenase, glutamate dehydrogenase, cholesterol dehydrogenase, aldehyde dehydrogenase, glucose dehydrogenase, fructose dehydrogenase, sorbitol dehydrogenase, lactate dehydrogenase, malic dehydrogenase, glycerol dehydrogenase, 17B hydroxysteroid dehydrogenase, estradiol 17B dehydrogenase, amino-acid dehydrogenase, glyceraldehydes 3-phosphate dehydrogenase, 3-hydroxysteroid dehydrogenase, diaphorase, catalase, peroxidase, glutathione reductase, NADH-cytochrome b5 reductase, NADPH-adrenoxin reductase, cytochrome b5 reductase, adrenodoxin reductase, and nitrate reductase.